Cryoprotective agent and its use in cryopreservation of cellular matter

Abstract

A novel class of nonpermeating cryoprotectants which, when mixed with certain known penetrating cryoprotectants, provide a useful medium for protection of living cells during a cryopreservation process. Algae-derived polysaccharides such as agarose and alginate are useful as nonpermeating cryoprotectants as they form a gel matrix when cooled, protect against ice crystal formation, and yield improved viability of cells when thawed.

What is claimed is: 
     
       1. A cryoprotective agent comprised of a mixture of a cell-permeating cryoprotectant and a non-cell permeating, macromolecular polysaccharide cryoprotectant derived from algae. 
     
     
       2. The cryopreservative agent of claim 1, wherein said nonpermeating cryoprotectant comprises a mixture of macromolecular polysaccharides. 
     
     
       3. The cryoprotective agent of claim 1 wherein said macromolecular polysaccharide is selected from the group consisting of agarose and alginate. 
     
     
       4. The cryoprotective agent of claim 1 wherein said cell-permeating cryoprotectant is selected from the group consisting of glycerol and dimethylsulfoxide. 
     
     
       5. The cryopreservative agent of claim 4, wherein said cell-permeating cryoprotectant is dimethylsulfoxide and said dimethylsulfoxide is present in a concentration from about 0.5M to 3.0M. 
     
     
       6. The cryopreservative agent of claim 5, wherein said dimethylsulfoxide is present in a concentration of about 1M. 
     
     
       7. A method for cryopreserving viable cellular matter comprising: (a) treating viable cellular matter with a cryoprotective agent comprising a mixture of a non-cell permeating cryoprotectant and a cell permeating cryoprotectant, said non-cell permeating cryoprotectant comprising an algae-derived macromolecular polysaccharide including from about 3 to about 3000 saccharide units such that said mixture gels at temperatures below ambient or when contacted by divalent ions, said non-cell permeating and cell permeating cryoprotectants being present in the mixture in concentrations effective for enhancing cellular viability upon transplant, and said non-cell permeating cryoprotectant being effective for encapsulating said cellular matter;   (b) freezing said encapsulated cellular matter and   (c) storing said frozen cellular matter.   
     
     
       8. The method of claim 7, wherein said nonpermeating cryoprotectant comprises a mixture of macromolecular polysaccharides. 
     
     
       9. A method of cryopreserving viable cellular matter comprising: treating viable cellular matter with a cryoprotective agent comprised of a cell-permeating cryoprotectant and a non-cell permeating, algae-derived macromolecular polysaccharide cryoprotectant, and   freezing the treated viable cellular matter to a temperature at least as low as the temperature of nitrogen vapor.   
     
     
       10. The method of claim 9, wherein said macromolecular polysaccharide is composed of a material selected from the group consisting of agarose and alginate. 
     
     
       11. The method of claim 9, wherein said cell-permeating cryoprotectant agent is selected from the group consisting of glycerol and dimethylsulfoxide. 
     
     
       12. The method of claim 11, wherein said dimethylsulfoxide is present in a concentration from about 0.5M to 3M. 
     
     
       13. The method of claim 12, wherein said dimethylsulfoxide is present in a concentration of about 1M. 
     
     
       14. The method of claim 9, wherein said cryopreservative agent comprises at least one algae-derived polysaccharide and dimethylsulfoxide. 
     
     
       15. The method of claim 9, wherein said cellular matter is selected from the group consisting of heart valves, corneas, pancreas, blood vessels, bone, bone marrow, endothelial cells, saphenous veins, liver cells, nerves, connective tissue endocrine glands and exocrine glands. 
     
     
       16. The method of claim 9 wherein said cellular matter is selected from the group consisting of ligaments, tendons and cartilage. 
     
     
       17. The method of claim 9 wherein said cellular matter is skin. 
     
     
       18. The method of claim 9 wherein said cellular matter is islets of Langerhans. 
     
     
       19. In a process for cryopreserving transplantable cellular matter wherein transplantable cellular matter is harvested from a donor and then cryopreserved at a temperature at least as low as the temperature of nitrogen vapor, the improvement comprising the step of encapsulating the transplantable cellular matter in an algae-derived polysaccharide gel prior to cryopreservation. 
     
     
       20. In a process for cryopreserving transplantable cellular matter wherein transplantable cellular matter is harvested from a donor and then cryopreserved at a temperature at least as low as the temperature of nitrogen vapor, the improvement comprising the steps of: (a) treating the cellular matter with a mixture of at least one algae-derived polysaccharide and dimethylsulfoxide in amounts which are effective for reducing ice crystal formation; and   (b) cooling said treated cellular matter such that a cell is formed around said treated cellular matter.   
     
     
       21. A cryopreservative agent comprising a mixture of a non-cell permeating cryoprotectant and a cell-permeating cryoprotectant, said non-cell permeating cryoprotectant comprising an algae-derived macromolecular polysaccharide having about 3 to about 3000 saccharide units,   said non-cell permeating and cell permeating cryoprotectants being present in the mixture at concentrations which are effective (a) for forming a gel at a temperature below ambient temperature or upon contact with divalent ions, and (b) improving the biological function of the transplantable, cryopreserved tissue upon transplant into a patient in need of such treatment.   
     
     
       22. A cryoprotective agent of claim 21 wherein the cell-permeating cryoprotectant is dimethylsulfoxide. 
     
     
       23. A method of improving the post-transplantable biological function of a transplantable tissue comprising: treating the transplantable tissue prior to cryopreservation with a mixture of a non-cell permeating cryoprotectant and a cell-permeating cryoprotectant,   said non-cell permeating cryoprotectant comprising an algae-derived macromolecular polysaccharide containing about 3 to about 3000 saccharide units per molecule,   said non-cell permeating and cell-permeating cryoprotectants being present in the mixture in concentrations which are effective (a) for forming a gel at a temperature below ambient temperature or upon contact with divalent ions, and (b) for improving the biological function of the transplantable, cryopreserved tissue upon transplant into a patient in need of such treatment.   
     
     
       24. A method in accordance with claim 23 further comprising: encapsulating the transplantable tissue in an algae derived polysaccharide, and   cryopreserving the encapsulated transplantable tissue at a temperature effective for improving the biological function of the tissue.   
     
     
       25. The method of cryopreserving viable cellular matter comprising: treating viable cellular matter with a mixture of at least one algae-derived macromolecular polysaccharide and dimethylsulfoxide in amounts which are effective for improving the biological function of said cellular matter upon transplant,   said algae-derived polysaccharide encapsulating said cellular matter, and   freezing said encapsulated cellular matter in said mixture.   
     
     
       26. A method of transplanting cellular matter from a donor into a recipient, comprising: (a) removing cellular matter from a donor;   (b) treating the cellular matter with a mixture of at least one algae-derived polysaccharide and dimethylsulfoxide in amounts which are effective for improving the biological function of the cellular matter upon subsequent thawing and implant, and for encapsulating said cellular matter with the algae-derived polysaccharide;   (c) freezing said encapsulated cellular matter;   (d) storing said frozen cellular matter;   (e) thawing said frozen cellular matter; and   (f) implanting the thawed cellular matter into the recipient.   
     
     
       27. A method for cryopreserving cellular matter comprising: (a) removing the cellular matter from a donor;   (b) forming an algae-derived polysaccharide gel around said cellular matter;   (c) adding at least one cell-penetrating cryoprotectant to said gel-enclosed cellular matter in an amount effective for reducing ice crystal formation during cryopreservation, and   (d) storing the cellular matter for a desired period of time.   
     
     
       28. A method of transplanting cellular matter from a donor to a recipient, comprising: (a) removing cellular matter from a donor;   (b) forming a algae-derived polysaccharide gel around said cellular matter;   (c) adding a cell-penetrating cryoprotectant to the gel-enclosed cellular matter in an amount effective for reducing ice crystal formation;   (d) freezing the gel-enclosed cellular matter and cell-penetrating cryoprotectant;   (e) storing the frozen cellular matter for a desired period of time;   (f) thawing said frozen cellular matter, and implanting the cellular matter in a recipient.